Cancer research has been revolutionized by the realization that inactivating somatic mutations in tumor suppressor genes are a key component that drives the process of tumorigenesis. While the discovery of such tumor suppressor genes have had profound implications for identification of at-risk individuals in rare cancer prone-families and have contributed to a better understanding of the biological mechanisms of tumor progression, thus far they have led to the discovery of few (if any) new drugs. Academic and pharmaceutical company researchers are currently grappling with this difficult question - what are the best ways to translate the discovery of tumor suppressor genes into therapeutics that target them? PTEN is a prototype tumor suppressor gene commonly inactivated by mutations in glioblastoma, endometrial cancer, melanoma, prostate cancer, and other tumor types. We have recently employed human somatic cell gene targeting to create isogenic sets of human HCT116 colon cancer cells that differ only in the presence or absence of their endogenous wild-type PTEN genes. Here we propose to employ this set of cells as the basis of a cell-based screen to identify small molecules that are specifically cytotoxic or cytostatic towards PTEN-deficient cells. Such compounds would be useful probes for study of the PTEN tumor suppressor pathway and might form the basis of therapeutics for treatment of cancers harboring mutations in PTEN. Furthermore, we propose to create additional isogenic sets of PTEN gene-targeted human cancer cells to function as secondary screens for confirmation of the hits obtained in the primary assay. Specific Aim #1: Validate and implement a cell-based screen for the identification of molecules that specifically kill PTEN-deficient human cancer cells. Specific Aim #2: Employ human somatic cell gene targeting to create additional isogenic sets of human cancer cells differing only in the presence or absence of PTEN. Validate the hits obtained in Aim #1 in these new cell lines. [unreadable] [unreadable]